Liposomes are microscopic closed vesicles having an internal phase enclosed by one or more lipid bilayers, and are capable of holding water-soluble material in the internal phase, and lipophilic material in the lipid bilayer. When entrapping an active compound in liposome, and delivering it to target tissue, how to entrap the active compound in the liposome with high efficiency, and how to secure stable retention of the active compound by the liposome constitute important issues.
When entrapping lipophilic compounds in liposome, a high entrapment ratio can be achieved relatively easily, but except in cases of compounds which have very high membrane affinity such as amphotericin B (the principal agent in the liposomal drug AmBisome), retention stability in blood plasma is ordinarily low, and it is difficult to obtain sufficient improvement in pharmacokinetics. With respect to methods for entrapping water-soluble compounds in liposome, there are various methods such as the lipid film method (Vortex method), reverse phase evaporation method, surfactant removal method, freeze-thaw method, and remote loading methods (pH gradient method, ion gradient method). However, it is only the remote loading methods that provide close to a 100% entrapment ratio; an entrapment ratio on the order of only 5 to 30% is obtained from the other methods.
As remote loading methods, those using a pH gradient and ammonium sulfate ion gradient are known. The pH gradient method, which is a remote loading method using a pH gradient, is a technique for incorporating compounds into liposome by using the movement of molecular/ionic dissociation equilibrium due to the pH of the target compound.
As one example of a compound entrapped in liposome by the pH gradient method, one may cite, for example, doxorubicin (DOX, pKa: 8.2). After preparing a liposome solution with a buffer solution of pH 4, the external phase of the liposome is replaced with a pH 7 buffer solution. In the case where DOX is added to this liposome solution, as the molecular DOX in the pH 7 solution is lipophilic, it migrates to the liposome membrane rather than to the aqueous phase. In the case where the DOX that has migrated to the liposome membrane further contacts the pH 4 internal phase of the liposome, it becomes ionic, and is incorporated into the internal phase of the liposome. In this way, DOX is transported from the external phase to the internal phase of liposome by a movement of dissociation equilibrium (see Non-patent Literature 1, Non-patent Literature 2, and Patent Literature 1).
A variety of techniques have been reported for improving this type of remote loading method.
In Non-patent Literature 3, a technique is disclosed for improving the entrapment ratio of active compounds by adding ethanol together with the active compound to the external phase of the liposome, when the pH gradient method is conducted in liposome of special composition called cholesterol-free liposome.
In Patent Literature 2, in addition to the pH gradient, a technique is disclosed for improving the entrapment ratio of active compounds by having copper ions exist in the internal phase of the liposome.
Instead of a pH gradient in the pH gradient method, the ammonium sulfate method, which is a remote loading method using an ammonium sulfate ion gradient, is a technique for incorporating active compounds into the internal phase of liposome by using an ion gradient of bivalent ammonium sulfate (see Non-patent Literature 1 and Patent Literature 3).
In addition to an ion gradient based on ammonium sulfate, Patent Literature 4 discloses a technique for incorporating active compounds into liposome by adding boronic acid together with the active compound to the external phase of the liposome.
Instead of an ion gradient based on ammonium sulfate, Patent Literature 5 discloses a technique wherein, compared to the case where ammonium sulfate is used, the release rate of the active compound is improved by incorporating the active compound into liposome using an ion gradient of glucuronic acid anion.
Thus, from the standpoint of entrapment ratio, remote loading methods are excellent entrapment methods. However, in the case where remote loading methods are used, except for special cases such as Doxil (a liposome preparation of DOX) where the active compound entrapped in the internal phase of the liposome is crystallized, there is the problem that the active compound tends to leak from the liposome in blood plasma, and that retention stability of the active compound is low.
On the other hand, a technique is disclosed for solubilizing in advance an active compound with cyclodextrin (hereinafter, also referred to as “CyD”) and then entrapping a complex of the cyclodextrin and the active compound into the liposome by a Vortex method or the like in order to enhance retention stability or enhance the solubility of an active compound. However, this method results in only an entrapment ratio of 5 to 20%, at which large-scale production is very difficult to perform.
In Non-patent Literature 4, a compound is used, in which salicylic acid is covalently bonded to CyD as a model compound, and a lipid film method is adopted for entrapping the compound into the liposome (an entrapment ratio is 8% or lower). It is suggested that the active compound forms a complex with CyD in the liposome internal phase and thus retention stability is enhanced.
In Non-patent Literature 5, a poorly water-soluble compound betamethasone is solubilized in advance with several types of CyD derivatives and entrapped into the liposome by a lipid film method (an entrapment ratio is 3% or lower). It is indicated that retention stability is enhanced and thus slow release effect is provided by the use of CyD derivatives having a high association constant with betamethasone.
Moreover, in Non-patent Literature 6, a poorly water-soluble compound ketoprofen is solubilized in advance with HP-β-cyclodextrin, and various entrapment methods have been attempted for the obtained complex. MLVs (multilamellar vesicles) achieve a relatively high entrapment ratio of approximately 75%, whereas SUVs (small unilamellar vesicles), which are used with the aim of EPR effect, remain at an entrapment ratio of approximately 55%. However, entrapment into the liposome internal phase with such a high entrapment ratio is theoretically impossible by the entrapment methods used in the document. Thus, the ketoprofen is highly likely to be distributed in the lipid bilayer, not in the liposome internal water phase.
Furthermore, Non-patent Literature 7 discloses that the liposome membrane permeability of a water-soluble substance is enhanced by creating in advance a complex of prednisolone and CyD and entrapping the complex into a liposome by a freeze-thaw method.
Non-patent Literature 8 discloses that the liposome entrapping the complex of DOX and γ-cyclodextrin exhibits a higher intratumoral DOX concentration and antitumor effect than those of the liposome entrapping only DOX. In said document as well, the complex of DOX and γ-cyclodextrin is formed in advance, and this complex is entrapped in the liposome. Likewise, in Patent Literature 6, a technique is disclosed for achieving slow release of an active compound by forming in advance the complex of the water-soluble compound and CyD and thus entrapping this complex into the liposome.
As described above, with conventional technical methods, the current situation is that it is difficult to achieve coexistence of a high entrapment ratio of the active compound in liposome and retention stability of the active compound in liposome.